Plant responses to genotoxic stress are linked to an ABA/salinity signaling pathway

The Plant Journal (1999) 17(1), 73–82

Plant responses to genotoxic stress are linked to anABA/salinity signaling pathway

Doris Albinsky1, Jean E. Masson†, Augustyn Bogucki1,

Karin Afsar1, Imre Vass2, Ferenc Nagy2 and

Jerzy Paszkowski1,*

1Friedrich Miescher-Institute, PO Box 2543,

CH-4002 Basel, Switzerland, and2Institute of Plant Biology, Biological Research Center,

H-6701 Szeged, PO Box 521, Hungary

Summary

Cells have developed a complex network of reactions to

avoid or reduce the deleterious consequences of DNA

damage. Responses to genotoxic stress include activation

of distinct stress signaling pathways, delay of cell cycle

progression and induction of DNA repair. In contrast to

other organisms, it is not known which signal transduction

pathways sense genotoxic stress in plants. Here we

describe an Arabidopsis mutant (uvs66) that appears to

be affected in the perception of signals triggered by

genotoxic treatments. The mutant uvs66 was identified

as hypersensitive to UV-C and to the DNA-damaging

chemicals methyl methane sulfonate (MMS) and mito-

mycin C (MMC), but seems to perform light dependent

repair, nucleotide excision repair and homologous recom-

binational repair as efficiently as the wild type. Exposure

of uvs66 plants to various environmental stresses revealed

a normal response, with the exception of elevated salinity

and abscisic acid (ABA). The hypersensitivity to NaCl and

ABA is correlated with aberrant regulation of transcripts

that are regulated by ABA (RAB18), or are induced by DNA

damaging treatments (AtRAD51). The properties of the

mutant uvs66 suggest an unexpected link between ABA

and/or salt stress mediated signals and genotoxic stress

responses, and provide an important connection between

the physiological and genetic responses of plants to abiotic

stress factors.

Introduction

The UV-B, which penetrates the cuticle and epidermal

tissue, has harmful effects on cell components such as DNA

and proteins. Cells respond to UV light by the activation of

genes in specific-stress response pathways (Fuglevand

et al., 1997; Green and Fluhr, 1995; Ohl et al., 1989) and by

the induction of DNA-repair activities (Puchta et al., 1995).

*For correspondence (fax 141 61 6973976; e-mail [email protected]).†Present address: INRA, Unite de Recherche Vigne et Vin, F-68000

Colmar, France.

© 1999 Blackwell Science Ltd 73

DNA damage caused by UV is repaired by several

mechanisms acting in parallel, in particular photorepair,

nucleotide excision repair (NER) and recombinational

repair. These pathways have been primarily elucidated in

bacteria, yeast and mammals (see for example, Wood,

1996). More recent attempts at a genetic dissection of plant

DNA repair components have resulted in the isolation of

several mutants hypersensitive to UV-light and γ- or X-ray

radiation (Britt et al., 1993; Davies et al., 1994; Harlow et al.,

1994; Jenkins et al., 1995; Jiang et al., 1997a; 1997b; Landry

et al., 1995; Landry et al., 1997; Li et al., 1993; Masson et al.,

1997). The cause of the UV hypersensitivity of some of

these mutants is not well defined. The better-characterized

mutants can be grouped into two classes: mutants defective

in the production of UV-absorbent flavonoid compounds

(Landry et al., 1995; Li et al., 1993), and mutants defective

in DNA repair. In the latter class, two genetic defects have

been characterized. Mutant uvr1 was found to be impaired

in the dark repair of 6–4 photoproducts (Britt et al., 1993)

and uvr2–1 is deficient in photolyase (Jiang et al., 1997a;

Landry et al., 1997).

In addition to DNA damage, UV light induces specific

cellular signals, and mutations in genes involved in such

signaling cascades may also reduce UV resistance. In

mammalian cells, the response to UV and other DNA-

damaging agents is manifested by the activation of tran-

scription factor AP-1, which consists of the two subunits,

c-fos and c-jun. The expression of c-fos has a protective

function in cells exposed to genotoxic agents, and cells

depleted of c-fos are hypersensitive to these agents

(Schreiber et al., 1995). It is not clear if DNA damage is

involved in AP-1 activation, since the UV triggers can be

transmitted from the cell surface to the nucleus by a MAP-

kinase cascade (Devary et al., 1993; Liu et al., 1996; Rosette

and Karin, 1996) and oxidative stress signals. In contrast

to mammals, plant cellular pathways responsible for a co-

ordinated reaction to genotoxic stresses are not known.

Thus, the recovery and characterization of mutants hyper-

sensitive to DNA-damaging treatments, but with unaltered

DNA-repair activities, could be of particular value in the

determination of such signaling cascades.

Here we report the isolation and characterization of the

Arabidopsis thaliana mutant uvs66 which is highly sensitive

to UV-light and DNA-damaging chemicals. However, uvs66

is probably neither impaired in basic DNA-repair processes

(photorepair, NER and homologous recombination) nor in

response to oxidative stress. Surprisingly, uvs66 exhibits

a hypersensitivity to abscisic acid (ABA) and NaCl. There-

fore, the uvs66 mutation provides an unexpected link

74 Doris Albinsky et al.

between genotoxic stress responses and a signaling path-

way regulated by ABA and/or salinity and suggests that

oxidative burst is not involved in these signals.

Results

Isolation of the UV hypersensitive mutant uvs66

The isolation was based on the root irradiation procedure

developed for the isolation of Arabidopsis mutants hyper-

sensitive to X-rays (Masson et al., 1997). Protection of

shoots during UV-C treatment allowed the rescue of hyper-

sensitive individuals. The screen was directed towards the

recovery of individuals affected in the dark repair of UV

damage. Thus, after UV irradiation, plants were kept in the

dark for 24 h. Dose–response experiments under these

conditions determined that 5 kJ m–2 applied to root tips

caused the termination of main root growth of the wild

type. Irradiation at lower doses provoked a temporary

reduction of growth followed by recovery after 24 h. The

dose of 3 kJ m–2 was chosen to screen for individuals with

increased UV sensitivity. Among 11 000 ethyl methane

sulfonate (EMS)-mutagenized M2 seedlings examined, 72

were selected as primary mutant candidates since growth

of their roots did not recover after UV treatment. UV-C

sensitivity was re-examined in their progeny (M3) and

19 lines were confirmed to exhibit a UV-hypersensitive

phenotype after irradiation with 3 kJ m–2. One line (uvs66)

showed sensitivity even at 0.75 kJ m–2. Importantly, the

uvs66 plants grown under standard phytotron or green-

house conditions were phenotypically indistinguishable

from the wild type. Therefore, UV-hypersensitivity and

specific alteration of stress responses described later are

not due to the overall weakness of the mutant.

For further studies, selfed progeny of uvs66 (M5) and

three independently recovered backcrossed lines homo-

zygous for the uvs66 allele were used in parallel assays.

Thus, the concomitant phenotypes described here are likely

to be specific for the uvs66 allele, although two very closely

linked genes cannot be excluded.

Sensitivity to DNA-damaging agents

To determine whether the mutant uvs66 was also sensitive

to DNA-damaging treatments other than UV, seedlings

were tested for their ability to grow in the presence of

DNA cross-linking and radio-mimicking agents, mitomycin

C (MMC) and methylmethane sulfonate (MMS), respec-

tively. The minimal lethal doses for wild-type seedlings

of Arabidopsis thaliana ecotype Landsberg erecta were

determined to be 20 mg l–1 and 200 p.p.m. for MMC and

MMS, respectively (Masson et al., 1997). Seedlings of the

uvs66 genotype died at 10 mg l–1 MMC and 100 p.p.m.

MMS. Exposure of the wild type to these doses had

© Blackwell Science Ltd, The Plant Journal, (1999), 17, 73–82

Figure 1. The comparison of (a) MMS and (b) UV-B sensitivity of the wild

type and uvs66.

(a) Five-days-old seedlings were transferred to media containing increasing

concentrations of MMS. The multi-vial plate was photographed 2 weeks

later.

(b) The main root growth of uvs66 and wild type after UV-B irradiation.

The main roots of pre-germinated seedlings grown on vertical plates

were irradiated at day 0. After irradiation, seedlings were exposed to

photoreactivation conditions before transfer to red light (j j j u j j j uvs66,j j j d j j j wild type) or shifted immediately to red light (—u— uvs66, —d—

wild type). Non-irradiated controls (j j u j j uvs66, j j d j j wild type).

no visible effect, whilst selfed and backcrossed uvs66

seedlings were arrested in growth and became chlorotic

within 1 week (Figure 1a).

Genetic analysis

Since chemical tests are easy and rapid, genetic analysis

was accomplished on the basis of the MMS and MMC

sensitivities. The mutant was backcrossed to the wild type

and the segregation of sensitivity trait in the F2 was

examined on media containing MMS or MMC. Of 130

seedlings, 96 were resistant and 34 were sensitive to MMS,

Signaling of genotoxic stress 75

and out of 95 seedlings, 79 were resistant and 16 were

sensitive to MMC. Both values were not significantly differ-

ent from a 3:1 segregation (X2 5 0.09 and 3.37, respectively,

at P ø 0.05), indicating that uvs66 is a single recessive trait.

In order to assign the uvs66 allele to a specific chromo-

some, the mutant was crossed to marker lines (NW) carry-

ing visible mutations allocated to different chromosomes

(Meyerowitz and Ma, 1994, see Experimental procedures).

Plants with wild-type phenotypes were selected in the F2

generation and grown to yield F3 single plant progenies.

These were first examined for the segregation of the visible

marker phenotypes. Single plant offspring segregating

these phenotypes were examined for the uvs66 mutation

(sensitivity to MMS). Assuming an independent segre-

gation, we expected one out of four F3 populations hetero-

zygous for the tester mutations to be homozygous for the

uvs66 mutation. This was confirmed in all but one line

containing markers on chromosome 2: no MMS sensitivity

was detected in 27 families tested. This suggests that the

uvs66 allele is linked to chromosome 2 in the vicinity of

visible markers. Phenotypic markers for chromosome 2

used in the test crosses were hy1–1 (elongated hypocotyl)

and as-1 (yellow asymmetric and lobed leaves) located at

43.8 cM and 59.9 cM, respectively.

Photoreactivation property

Photorepair is the most important, light-dependent reaction

reverting UV damage in DNA. The photorepair properties

of mutant uvs66 were examined by applying 2.9 kJ m–2 of

UV-B light to the roots followed by transfer, either to

red light (660 nm, which allows photosynthesis but no

photoreactivation), or for 90 min to blue, photoreactivating

light. Seedlings from both treatments were grown for 4

subsequent days under red-light conditions. The main

roots of the wild-type seedlings grew independently of

photoreactivation. In contrast, the main root of uvs66

continued growing only after photoreactivation (Figure 1b).

Thus, uvs66 is proficient in photorepair and its UV hyper-

sensitivity results from defects in light-independent pro-

cesses.

Dark repair of DNA damage

The hypersensitivity of uvs66 to UV-B and UV-C, accom-

panied by increased sensitivity to MMS and MMC but

with intact photorepair, suggests a deficiency in DNA dark-

repair reactions such as NER or recombinational repair. To

test this hypothesis, a transient dark repair assay of UV-C-

irradiated plasmid was used (for details see Experimental

procedures). Under these conditions UV damage should

be repaired mainly by NER. UV-treated DNA of plasmid

pGUS23 carrying as a marker the hybrid β-glucuronidase

gene (Figure 2a) was introduced into protoplasts of the


wild type and uvs66 and repair/expression was allowed

for 24 h in the dark. Irradiation of the DNA template with

increasing UV doses caused decreased gene expression as

measured by accumulation of the β-glucuronidase specific

stain (Figure 2b). This was expected as a result of plasmid

template damage. At a dose of 5 kJ m–2, gene expression

was reduced to 20% of the unirradiated control. Import-

antly, the relationship of reduction to increasing radiation

doses did not differ significantly between uvs66 and the

wild type, which suggests a similar activity of damaged

templates in both lines, and thus the likely similar rates of

their repair.

UV-light, MMC and MMS are potent inducers of intra-

chromosomal recombination in plants (Lebel et al., 1993;

Puchta et al., 1995). Yeast mutants affected in recombina-

tional repair are often hypersensitive to MMS (Prakash and

Prakash, 1977). We have determined the proficiency of

the mutant in homologous recombination. Two different

homologous recombination mechanisms are described for

Saccharomyces cerevisiae (Klein, 1988; Prado and Aguilera,

1995). DSB situated within regions of homology can be

repaired through the RAD52 pathway, whereas DSB

located outside homologous regions require the action of

the RAD1 and RAD10 complex for endonucleolytic removal

of the non-homologous flank (Tomkinson et al., 1993).

Therefore, uvs66 and wild-type cells were compared in

two corresponding recombination assays. A pair of deletion

derivatives of plasmid pGUS23 (Puchta and Hohn, 1991;

Figure 2a) was co-transformed into protoplasts isolated

from uvs66 and the wild type. GUS expression required

restoration of a functional gene by homologous recom-

bination within the GUS coding region between two co-

transformed molecules. The homologous region was

located either at the end of linear DNA or was surrounded

by a non-homologous DNA stretch (Figure 2a). For both

types of recombination substrates, the mutant uvs66

showed recombination properties similar to the wild type

(Figure 2c).

Oxidative stress responses

Sensitivity of uvs66 to UV-light and DNA-damaging

agents could also be the result of hypersensitivity to stress

mediated by reactive oxygen species (ROS). To test this,

two independent assays were performed: (1) the measure-

ment of photosystem II (PSII) activity after UV exposure;

and (2) the test of sensitivity of the mutant seedlings to an

inducer or a scavenger of ROS.

The main target for UV-light-induced damage in plants

is the D1-protein of the reaction center of PSII (Aro et al.,

1993). We compared uvs66 and the wild type for reduced

photosynthetic activity of PSII after UV-B exposure. The

maximal photosynthetic efficiency of PSII was determined

by calculating the ratio of the variable and the maximal


fluorescence yields (Fvar/Fmax) (Dau, 1994) (values in

Figure 3a). UV-B-induced damage was also calculated from

the quantum yield of the entire photosynthetic electron

transport, according to Genty et al. (1989). There was no

significant difference in PSII activity between wild type and

mutant plants (Figure 3a), indicating the lack of an effect

of the uvs66 mutation on reactions to ROS.

This conclusion was supported by assaying sensitivity

to oxidative damage using increasing concentrations

of a reagent which directly induces ROS by forming

singlet oxygen (4,5,6,7,-tetrachloro-29,49,59,79-tetraiodo-

fluorescein–rose bengal) or of a scavenger of ROS (N-


acetyl-I-cysteine, NAC). The sensitivity of uvs66 to both

agents was not different to that of wild-type seedlings

(Figure 3b).

uvs66 is hypersensitive to salinity stress and ABA

The not drastically altered DNA repair and reaction to

oxidative damage suggested that the uvs66 hyper-

sensitivity to genotoxic treatments probably does not result

from a deficiency to cope with the injury, but rather from

failure to activate other cellular responses. To determine

whether signaling of genotoxic stress converges with

known cellular stress signaling pathways, we analyzed

responses of uvs66 to several adverse conditions (details

in Experimental procedures). Uvs66 was as resistant as

the wild type to elevated temperature, osmotic stress

and ethylene (data not shown), although a significant

hypersensitivity was found to increased salinity (Figure 4).

Figure 2. DNA dark-repair assays.

(a) Plasmid constructs used for the NER and homologous recombination

assays. Plasmid pGUS23 (Puchta and Hohn, 1991) containing the 35S

promoter of Cauliflower Mosaic Virus (hatched box) linked to the coding

region of the β-glucuronidase gene (open box) and the nopaline synthase

polyadenylation signal (black box). Restriction enzymes used for the

linearization of plasmid DNA prior to the transformation are indicated.

Plasmid pGUS23N1 is a 59 deletion derivative of pGUS23, and pGUS23C1

a 39 deletion derivative of pGUS23.

(b) Excision repair assay. β-glucuronidase gene expression after irradia-

tion of pGUS23 plasmid template with increasing doses of UV.

(c) Recombinational repair assay. Mesophyll protoplasts of uvs66 and the

wild type were transformed with (1) pGUS23 linearized with AatII or (2) a

mixture of pGUS23N1 cut with SnaBI and pGUS23C1 cut with BstB1 (DSB

proximal to homology), or (3) a mixture of pGUS23N1 cut with AatII and

pGUS23C1 cut with ScaI (DSB distal to homology). The β-glucuronidase

activity was normalized to luciferase activity used as an internal transforma-

tion control.


Salt sensitivity was linked to MMS sensitivity in three

independent backcrosses, indicating a direct connection of

this trait with the altered responses to DNA-damaging

treatments. Since there was no general hypersensitivity of

uvs66 to osmotic stress, a toxicity of Na1 or Cl– ions could

be envisaged. Responses of uvs66 and the wild type to

KCl did not differ (data not shown), and therefore increased

levels of Na1 ions have to be responsible for sensitivity of

uvs66 to NaCl. This phenotype is similar to that of the sos1

Figure 3. The comparison of wild type and uvs66 sensitivity to oxidative

stress.

(a) Photosystem II activity measured as a relative decrease in variable

chlorophyll fluorescence. Plants were irradiated for a maximal period of

5 h, corresponding to a dose of 280 kJ m–2. Wild type d, uvs66 u.

(b) Seedling sensitivity assay to Rose Bengal and N-acetyl cysteine (NAC)

(assay conditions similar to that of MMS sensitivity, Figure 1a).


mutation, which is also located on chromosome two (Wu

et al., 1996). However, sos1 has a wild-type resistance to

DNA damaging treatments (UV, MMS – data not shown)

suggesting alteration of different genes in both mutants.

The involvement of ABA in the mediation of salinity

stress signals has been discussed previously (Bostock and

Quatrano, 1992). Therefore, we have examined responses

of uvs66 to ABA. Two tests were used: (1) determination

of the minimal dose of ABA inhibiting development of pre-

germinated seedlings; and (2) main root growth retardation

by ABA. Uvs66 showed increased sensitivity to ABA in

both tests. The development of seedlings was inhibited by

the presence of 1 µM ABA to an extent visible in the

wild type only at 15 µM (data not shown). In addition, an

inhibitory effect of ABA was quantified by the determina-

tion of root growth retardation (Figure 5). ABA at 0.5 µM

had a drastic inhibitory effect on uvs66 root development.

uvs66 is affected in the regulation of the expression of

the RAB18 gene and the RAD51 homologue under

salinity stress

The RAB18 (responsive to ABA) gene codes for a serine-

and lysine-rich protein, and its mRNA accumulates in

response to dehydration and low temperature (but not to

elevated temperature) and ABA. RAB18 mRNA accumula-

tion is suppressed in ABA-insensitive mutants (abi1 and

abi2) (Gosti et al., 1995). We examined this regulation in

the genetic background of uvs66. Induction of RAB18 by

ABA was not altered in uvs66 (Figure 6a). Surprisingly,

although this gene was not, or was only slightly, activated

by salt stress in the wild type or ABA insensitive mutants,

it was clearly induced in uvs66 under salt stress (Figure 6b).

In addition, salt stress of uvs66 induced expression of a

gene homologous to RAD51. In the wild type, the RAD51

transcript accumulated to high levels in response to geno-

toxic treatments, such as X- or γ-radiations (Doutriaux

et al., 1998), but its accumulation was not stimulated by

salt stress (Figure 6b). In contrast to the wild type, the

RAD51 transcript in uvs66 accumulated in response to salt-

induced stress (Figure 6b). This is further evidence for a

link between signals triggered by ABA and/or salinity, and

genotoxic stress responses, i.e. the gene product UVS66

is apparently involved in both signaling pathways.

Discussion

The phenotype of uvs66 is consistent with a defect in a

light-independent DNA-repair activity or with defects in

other cellular responses to genotoxic stress. In the case of

DNA-repair deficiencies, sensitivity to UV followed by the

dark recovery and MMC suggest an impairment of the NER

pathway since the lesions, cyclobutane pyrimidine dimers

and 6–4 photoproducts induced by UV (Mitchell et al.,


Figure 4. Salt tolerance of uvs66.

Five-day-old seedlings were transferred to

medium supplemented with NaCl at the concen-

trations indicated. The picture was taken after

2 weeks further growth.

1991), together with the interstrand crosslinks induced by

MMC (Szybalski and Iyer, 1964), can be removed by NER.

Sensitivity to MMS, a radio-mimicking agent, may be the

result of defects in recombinational repair. Since we were

not able to link the uvs66 mutation to any of the main light-

independent DNA-repair pathways, we conclude either that

the DNA repair was not drastically affected or that the

applied assays were not sufficiently sensitive. The extra-

chromosomal homologous recombination assay has been

used successfully previously in studies of recombination

substrate preferences of plant cells (Baur et al., 1990;

Bilang et al., 1992; Puchta and Hohn, 1991) and for the

characterization of genetic deficiencies (Masson and

Paszkowski, 1997). The NER assay was developed and

used here for the first time, but the UV dose-dependent

reduction of template activity and its time-dependent

recovery in a cellular environment were in agreement with

the expectation of assay performance. Therefore, we favor

the conclusion that uvs66 is not deficient in these DNA-

repair activities.

The combined sensitivity towards three independent

genotoxic agents (UV, MMS, MMC), accompanied with the

possible proficiency of DNA-damage repair, implies that

uvs66 is affected either in the cellular responses down-

stream of the DNA damage or in a cellular signaling

pathway linking activities of repair of DNA damage and

other cellular stress responses. In answer to selected

stresses, such as high temperature and increased salinity,

plants react with an induction of chromosomal rearrange-

ments, measured as elevated levels of intrachromosomal

recombination (Lebel et al., 1993; Puchta et al., 1995).

Furthermore, increased temperature provokes an UV-

hypersensitive phenotype in Arabidopsis (Jenkins et al.,

1995). Since these challenges are rather unlikely to induce

DNA damage directly, the activation of a common inter-

mediate(s) in signaling cascades, linking physiological and

genetic responses, can be envisaged. In order to investigate


the involvement of uvs66 in stress responses, mutant and

wild type were subjected to a set of challenges known to

activate discrete signaling cascades in plants. For example,

the ROS is considered to be the critical signal in the

regulation of programmed cell death (PCD) and/or the

responses to pathogen attack (Pennel and Lamb, 1997).

The treatments with UV, MMC and/or the resulting DNA

damage in the uvs66 background may have interfered with

the ROS signals, leading to premature activation of PCD,

as manifested by the sensitivity of the mutant. However,

the unaltered sensitivity of uvs66 to direct induction of

oxidative stress and the application of ROS scavengers

suggests that this is unlikely. An unaltered degree of UV-

mediated oxidative damage in uvs66 was also demon-

strated by unchanged photosystem II activity after UV-B

irradiation (Aro et al., 1993). These results also show that

the penetration of UV-B light into leaves was unchanged.

All these observations are consistent with unaltered cellular

signals mediated by ROS.

Comparing responses of uvs66 and wild type to a series

of abiotic stresses and to increasing concentrations of ABA

or ethylene, hormonal mediators of abiotic stress signals

in plants, the mutant had a wild-type sensitivity level to

increased temperature, osmotic pressure, oxygen deple-

tion and increased ethylene, but was clearly inhibited by

elevated levels of NaCl and ABA. Furthermore, under

increased salinity, the mutant showed abnormal regulation

of RAB18, a stress- and ABA-regulated gene (Gosti et al.,

1995) and of the Arabidopsis homologue of RAD51, the

DNA strand-exchange protein involved in recombinational

repair. The steady-state level of RAD51 mRNA has been

shown to increase in response to DNA damage in yeast

(Basile et al., 1992) and Arabidopsis (Doutriaux et al., 1998).

In contrast, RAD51 transcript levels do not rise signi-

ficantly under salinity stress. This clearly differs in uvs66,

where increased salinity induces the accumulation of the

RAD51 transcript. Interestingly, although uvs66 is hyper-


Figure 5. Main root growth at increasing ABA concentrations.

Pre-germinated seedlings were transferred to vertical plates containing

ABA at the following concentrations: control (a), 0.5 µM (b), 1.0 µM (c)

and 3.0 µM (d) (u uvs66, d wild type). For every value, 10 independent

measurements were performed. Standard deviations of the means did not

exceed the diameters of the data points.

sensitive to ABA, the levels of both transcripts were norm-

ally regulated in response to the hormone. This illustrates

the complex interactions of ABA-dependent and ABA-

independent stress signaling pathways (Bostock and

Quatrano, 1992; Ishitani et al., 1997; Moons et al., 1997).

The novel genetic link discovered in the uvs66 mutant

between hypersensitivity to genotoxic agents and aberrant


Figure 6. Regulation of transcript levels under elevated exogenous ABA (a)

or under salt stress (b).

Steady state mRNA levels of RAB18 and RAD51 in uvs66 after ABA or salt

treatment at the concentrations indicated above blots (compare Experi-

mental procedures). In addition to wild type and uvs66, the ABA insensitive

mutants abi1 and abi2 (Leung et al., 1997) were included in these experi-

ments. The same filters were probed with RAB18, AtRAD51 (a), and RAB18,

AtRAD51 and 25S ribosomal RNA specific probes (b).

responses to NaCl and ABA is intriguing and provides

evidence for the convergence of ABA and/or NaCl mediated

signaling of selected abiotic stresses with genomic

responses. Since abiotic environmental challenges are

considered to be the major selective force in plant

evolution (Bohnert et al., 1995), such a link between percep-

tion of this force and genomic responses could be an

important factor, allowing for a fast and flexible genetic

adaptation of plant populations to a changing environment

(Schneeberger and Cullis, 1991; Walbot and Cullis, 1985).

Experimental procedures

Mutant screening

M2 populations of EMS mutagenized seed stocks of Arabidopsis

thaliana, ecotype Landsberg erecta, were provided by E. Grill, ETH

Zurich, Switzerland. Ten seeds per plate were sown in a row on

square plates (Sterilin) containing 60 ml of germination medium

(Masson and Paszkowski, 1992). The plates were kept for 2 days

at 4°C and then transferred to the growth chamber with 16 h light

at 25 µE m–2 s–1 (Osram Natura de Luxe) and 22.5°C and 8 h dark

at 22.5°C (Masson et al., 1997). Plates with germinating seedlings

were placed vertically in shielded boxes only allowing light

penetration from above. After the fifth day of sowing, the shoots

of the plantlets were protected by a plexiglas-cover, and the roots


irradiated with sublethal doses of UV-C light (3 kJ m–2, or 0.75 and

3 kJ m–2 for the re-screen) using a UV-C lamp with a fluence rate

of 30 W m–2 (Osram HNS 55 W ORF). Directly after irradiation,

plates were kept for 1 day in the dark or transferred directly to

light. The growth of the main roots of the seedlings was monitored

daily up to day 7.

Determination of the sensitivity to DNA-damaging

chemicals

The MMS (Sigma) and MMC (Fluka) sensitivity tests were carried

out using pre-germinated seedlings in a liquid germination

medium as described previously (Masson et al., 1997). Determina-

tion of the lethal doses for wild-type and mutant seedlings was

carried out at increasing concentrations of MMC (2.5, 5, 10, 15

and 20 mg ml–1) and MMS (25, 50, 100 and 150 p.p.m.). The

minimal discriminating doses (lethal to the mutant and tolerated

by the wild type) were 10 mg ml–1 MMC and 100 p.p.m. MMS.

Genetic analysis

The segregation analysis was performed on F2 segregating

populations after backcrossing to the wild type. Five-day-old F2

seedlings were either subjected to discriminating doses of MMC

and MMS, or the main roots were irradiated with sublethal doses

of UV-C light. Results of the segregation were analyzed by the

Chi-square test at P , 0.05. Randomly chosen F2 plants were

grown to maturity and their genotypes determined by segrega-

tion analysis of their progeny on DNA-damaging agents. Three

independent backcrossed uvs66 lines were selected for further

parallel characterization of physiological responses. For mapping,

NW-marker lines were provided by the Arabidopsis Seed Stock

Center, Nottingham, UK.

Photoreactivation assay

Seedlings were grown as for mutant screening and the root tips

were irradiated with 2.9 kJ m–2 of UV-B light using a Philips

TL40 W 1–2 UV-B lamp at a fluence rate of 6.12 W m–2. For photo-

reactivation, seedlings were treated for 90 min with blue light at

λ max 370 nm (fluence rate 5.8 W m–2) with a 331 nm cut-off filter.

Red light was provided at λ max 660 nm (fluence rate 6.7 W m–2)

for 4 days to UV-B-treated and control samples. Evaluation of root

growth was performed as described above.

Nucleotide excision repair and extrachromosomal

recombination assays

Assays were carried out with the marker plasmid pGUS23 coding

for the β-glucuronidase-gene (Puchta and Hohn, 1991; Figure 2a).

For the NER-assay, circular plasmid DNA was irradiated with

increasing doses of UV-C from 0 to 10 kJ m–2 using UV-cross-

linker 312 (Appligene). Five mg of UV-C-irradiated circular plasmid

DNA were transformed to 5 3 105 protoplasts of wild-type and

mutant plants, together with 5 µg of plasmid pGN35S-luc1 coding

for the firefly luciferase (LUC) (Gosti et al., 1995). The expression

of pGN35S-luc1 was not influenced by the radiation dose applied

to co-transformed pGUS23 plasmid, and stayed uniform through-

out all samples. The same amount of protoplasts was used as a

mock control. Mesophyll protoplasts were prepared and trans-

formed according to Damm and Willmitzer (1988) and Karesch

et al., 1991a,1991b). The growth conditions of the donor plants


and protoplast cultures were modified according to Masson and

Paszkowski (1992). After transformation, protoplasts were washed

and incubated for 24 h in the dark at 26°C in 2 ml of protoplast

plating medium (M1 medium; Masson and Paszkowski, 1992) at

a density of 7.5 3 104.

For the extrachromosomal recombination assay, two non-over-

lapping deletion derivatives of pGUS23 were used. The plasmid

pGUS23 linearized with Aat II was transformed to parallel samples

as a control. As an internal reference for transformation efficiency,

the plasmid pGN35S-luc1 was added to all samples. Transforma-

tion samples consisted of 7.5 3 105 protoplasts and 5 µg of each

plasmid DNA. The β-glucuronidase and luciferase assays were

performed after incubation for 24 h.

β-glucuronidase (GUS) and luciferase-(LUC) assays

For the GUS activity assay, the protoplast suspension was washed

twice by sedimentation for 5 min at 80 g in 8 ml of W5 solution.

Extraction buffer (100 ml) (50 mM Na2HPO4, 10 mM Na2EDTA, 0.1%

w/v Sarcosyl, 0.07% v/v β mercapto-ethanol, pH 7) was added to

the protoplast pellet. After brief mixing, the protoplast suspension

was frozen in liquid nitrogen. On thawing, the homogenate was

centrifuged at 15 800 g and the protein concentration in the

supernatant was measured according to Bradford (1976). For GUS-

activity, 17 µl of supernatant was mixed with 83 µl of 1 mM 4-

methyl-umbelliferyl-glucoronide (Sigma) dissolved in extraction

buffer. The mix was incubated at 37°C. At time 0 and after 1 and

24 h, aliquots of 20 µl were taken and the reaction was stopped

by the addition of 200 µl of 0.2 ml Na2CO3. The fluorescence was

determined with a GUS-Titertek Fluoroskan II (Flow Laboratories).

A dilution series of 4-methyl-umbelliferone (17.6 mg dissolved in

1 ml DMSO) was used as a standard. The GUS activity was

normalized for the protein concentration and luciferase activity

(Jefferson et al., 1987).

For the LUC activity assay, Luciferin (Promega) was mixed with

the protoplast suspension (2.8 3 104 cells) at a final concentration

of 1 mM. The bioluminescence (luciferase activity) of the protoplast

suspension was measured in a Micro Luminat LB 96 P (EG and

G. Berthold).

Activity measurements of PS II

Photosynthetic activity of the PS II was monitored by variable

chlorophyll fluorescence using a PAM fluorescence measuring

system (Walz, Effeltrich). The surfaces of wild-type and mutant

leaves were UV-B irradiated using a Vilbert-Lourmat lamp (fluence

rate 16 W m–2, λ max. 312 nm). UV-C was filtered out by 0.1 mm

cellulose acetate filter (Clairfoil, Courtaulds Chemicals). After UV

irradiation for 5 h (280 kJ m–2), plants were dark adapted for

10 min. The Fv/Fmax fluorescence parameter (i.e. ratio of the

variable and maximal fluorescence yield induced by saturating

light intensity) was used as a measure of the efficiency of the

photosynthetic activity. Measurements were repeated several

times, always using the same area of leaf surface to obtain the

time and dose-dependence of the UV-B damage.

Determination of stress tolerance (oxidative, osmotic,

salinity and high temperature)

Single pre-germinated seedlings (5 days old) were transferred to

multi-vial plates containing 1 ml of germination medium supple-

mented with increasing concentrations of stress-provoking com-


pounds and grown under standard conditions. The minimal lethal

dose of the different agents for the wild-type seedlings was

determined and used as the maximal concentration in the

sensitivity test: (1) rose bengal: 0, 0.1, 0.5, 1, 2 and 4 µM; or N-

acetylcysteine: 0, 0.3, 1, 3, 6 and 12 mM, (2) mannitol: 0, 0.1, 0.2,

0.4, 0.8, 1.0 M; (3) NaCl, KCl: 0, 0.04, 0.08, 0.12 M (the osmotic

pressure of all salt concentrations was determined and an equal

osmoticum of mannitol solution was used as an additional

control). Moving plates to 32°C for different time periods (1, 3

and 7 days) followed by standard growth conditions tested the

influence of temperature stress. Ethylene was applied in concentra-

tions of 10 p.p.m. for 5 days. The phenotypes of wild type and

uvs66 on the ABA-containing media were compared after 1 and

2 weeks of growth.

ABA-sensitivity assays

Sensitivity to ABA was determined in two tests. First, pre-

germinated, 5-day-old seedlings were transferred into multi-vial

plates containing liquid germination medium with increasing

concentrations of ABA (0, 0.5, 1, 3, 5, 10, 15 µM). The phenotypes

of wild type and uvs66 on the ABA containing media were

compared after 2 weeks of growth. A second test was performed

on vertical plates with different concentrations of ABA in the

medium (0, 0.5, 1, 3 µM) and ABA-mediated inhibition of the main

root growth was determined.

Northern blots

One-week-old seedlings were transferred for 24 h to medium with

increasing ABA, or NaCl concentrations. Incubation was followed

by freezing in liquid nitrogen and RNA extraction (Gosti et al.,

1995). Total leaf RNA (10 µg) was subjected to gel electrophoresis

in a 1.2% agarose gel and transferred to Hybond N (Amersham)

filters, which were hybridized for 18 h at 45°C in a solution

containing 5 3 SSC, 5 3 Denhardt’s, 10 mM Pipes, pH 6.4, 5 mM

EDTA, 50% formamide, 0.2 mg ml–1 of sheared salmon sperm DNA

and 0.1 mg ml–1 heparin. The radioactive probes were prepared by

the random primer method (Maniatis et al., 1989). Filters were

washed twice for 30 min each in 0.1 3 SSC, 0.1% SDS, 10 mM

sodium phosphate, pH 7.5 at 65°C and exposed to X-ray film

(Kodak).

Acknowledgements

We thank Barbara Hohn, Fred Meins, Katia Revenkova and Ortrun

Mittelsten Scheid for helpful suggestions during preparation of

the manuscript. We also thank Marie-Pascal Doutriaux and

Charles White for providing us with the AtRAD51 clone prior to

a publication, and A. Batschauer and J. Felix for their help

in photoreactivation experiments and ethylene treatments,

respectively.

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Plant responses to genotoxic stress are linked to an ABA/salinity signaling pathway